1. Field of the Invention
The present invention relates to a method of inhibiting cytochrome P450 enzymes by administering a Tripterygium Wilfordii Hook. F. (TW) extract. More specifically, inhibitions of cytochrome P450 (CYP450) isoforms 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 in cryopreserved human hepatocytes were studied. The method is particularly useful in determining drug-drug interactions when the TW extract is co-administered with other drugs.
2. Description of the Related Art
Cytochrome P-450 is a superfamily of enzymes that metabolize a large number of drugs, xenobiotics and endogenous substances in vitro and in vivo. Enzymes of the cytochrome P450 superfamily catalyze the oxidative metabolism of a variety of substrates, including natural compounds such as steroids, fatty acids, prostaglandins, leukotrienes, and vitamins, as well as drugs, carcinogens, mutagens, and xenobiotics. Cytochrome P450s, also known as P450 heme-thiolate proteins, usually act as terminal oxidases in multi-component electron transfer chains, called P450-containing monooxygenase systems. Specific reactions catalyzed include hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S-, and O-dealkylations, desulfation, deamination, and reduction of azo, nitro, and N-oxide groups. These reactions are involved in steroidogenesis of glucocorticoids, cortisols, estrogens, and androgens in animals; insecticide resistance in insects; herbicide resistance and flower coloring in plants; and environmental bioremediation by microorganisms. Cytochrome P450 actions on drugs, carcinogens, mutagens, and xenobiotics can result in detoxification or in conversion of the substance to a more toxic product. Cytochrome P450s are abundant in the liver, but also occur in other tissues. Members of the cytochrome P450 family are present in varying levels and their expression and activities are controlled by variables such as chemical environment, sex, developmental stage, nutrition and age.
More than 200 cytochrome P450 genes have been identified. There are multiple forms of these P450 and each of the individual forms exhibit degrees of specificity towards individual chemicals in the above classes of compounds. In some cases, a substrate, whether a drug or a carcinogen, is metabolized by more then one of the cytochromes P450. All cytochrome P450s use a heme cofactor and share structural attributes. Most cytochrome P450s are 400 to 530 amino acids in length. The secondary structure of the enzyme is about 70% alpha-helical and about 22% beta-sheet.
Genetic polymorphisms of cytochromes P450 result in phenotypically-distinct subpopulations that differ in their ability to perform biotransformations of particular drugs and other chemical compounds. These phenotypic distinctions have important implications for selection of drugs. For example, a drug that is safe when administered to most humans may cause toxic side-effects in an individual suffering from a defect in an enzyme required for detoxification of the drug. Alternatively, a drug that is effective in most humans may be ineffective in a particular subpopulation because of lack of a enzyme required for conversion of the drug to a metabolically active form. Further, individuals lacking a biotransformation enzyme are often susceptible to cancers from environmental chemicals due to inability to detoxify the chemicals.
Human cytochrome P450 1A2 constitutes about 13% of total P450 in human liver and is the second most abundant P450 following human cytochrome P450 3A4. P450 1A2 catalyzes the metabolism of a large variety of drugs and carcinogens. Drugs metabolized by human P450 1A2 include phenacetin, R-warfarin, clomipramine, imipramine, theophyline, theobromine, paraxanthine, caffeine, chlorzoxazone, 7-methoxyresorufin, and 7-ethoxycoumarin. P450 1A2 also has a major role in activating mutagens and carcinogens. For example, 1A2 metabolically activates the food pyrolysis products IQ and MeIQx to active mutagens.
A complication in patient drug choice is that most drugs have not been characterized for their metabolism by P450 1A2 and other cytochromes P450. Without knowing which cytochrome(s) p450 is/are responsible for metabolizing an individual drug, an assessment cannot be made for the adequacy of a patient's P450 profile. For such drugs, there is a risk of adverse effects if the drugs are administered to deficient metabolizers.
The cytochrome P-450 3A (CYP 3A) isoenzyme is a member of the cytochrome P-450 superfamily. It constitutes up to 60% of the total human liver microsomal cytochrome P-450 and is responsible for metabolism of a large number of drugs including nifedipine, macrofide antibiotics including erythromycin and troleandomycin, cyclosporin, FK506, teffenadine, tamoxifen, lidocaine, midazolam, triazolam, dapsone, diltiazem, lovastatin, quinidine, ethylestradiol, testosterone, and alfentanil. In addition, CYP 3A has been shown to be involved in both bioactivation and detoxication pathways for several carcinogens in vitro.
The active form of CYP 3A has been found in other organs besides the liver including kidney epithelial cells, jejunal mucosa, and the lungs. In these organs, the amount of the cytochrome P450 protein is much lower then in the liver. In a study of human lung microsomes, presence and activity of CYP 3A has been demonstrated.
Presence of the cytochrome P-450 3A in the lung microsomes indicates that the drugs and other substances which are subject to CYP 3A (P450-3A) mediated metabolism may be partially metabolized in the lungs. This has been demonstrated for the topical steroid, beclomethasome dipropionate. It has also been shown that only about 10% of the drug released by an inhaler is available to the lungs. The remaining mount is retained in the spacer device and oral cavity. Steroids absorbed from the lungs and gastrointestinal tract are subsequently metabolized by hepatic cytochrome P450. Many drugs, such as prednisone, cyclosporin, cyclophosphamide, digitoxin, diazepam, ethinylestradiol, midazolam, triazolo-benzodiazepines, dihydropyridine calcium channel blockers, certain HMG-CoA reductase inhibitors, etc. are metabolized by a member of the CYP3A family, CYP3A4.
Human CYP2A6 is an important member of the CYP superfamily and is present in liver up to 1% of the total CYP content (Yun et al., 1991). Human CYP2A6 metabolically activates the carcinogens aflatoxin B1 (Yun et al., 1991), a tobacco-specific nitrosamine 4-methylnitrosamino)-1-(3-pyridyl)-1-butone (Crespi et al., 1991), and N-nitrosodiethylamine (Fernandez-Salguero & Gonzalez, 1995). CYP2A6 also carries out coumarin metabolism by aromatic hydroxylation in humans (Pearce et al., 1992). Coumarin 7-hydroxylation has been used as a marker for CYP2A6 activity in vitro (Yamano et al., 1990) and the basis for measuring the in vivo expression of CYP2A6 (Cholerton et al., 1992; Rautio et al., 1992). A genetic polymorphism has been found in CYP2A6 (Fernandez-Salguero et al., 1995) that is due to three variant allelic forms, i.e., CYP2A6*1, 2A6*2, 2A6*3, respectively (Daly et al., 1996).
Cytochrome P450 2D6, also known as debrisoquine hydroxylase, is the best characterized polymorphic P450 in the human population (Gonzalez et al., Nature 331:442–446 (1988)). A poor metabolizer phenotype has been reported which behaves as an autosomat recessive trait with an incidence between 5 and 10% in the white population of North America and Europe. Poor metabolizers exhibit negligible amounts of cytochrome P450 2D6 (Gonzales et al., supra). Genetic differences in cytochrome P450 2D6 may be associated with increased risk of developing environmental and occupational based diseases. See Gonzalez & Gelboin, J. Toxicology and Environmental Health 40, 289–308 (1993)).
There is some evidence that S-mephenytoin 4′ hydroxylase activity resides in the cytochrome P450 2C family of enzymes. A number of 2C human variants (designated 2C8, 2C9 and 2C10) have been partially purified, and/or cloned. A comparison of the P450 2C cDNAs and their predicted amino acid sequences shows that about 70% of the amino acids are absolutely conserved among the human P450 2C subfamily. Some regions of human P450 2C protein sequences have particularly highly conservation, and these regions may participate in common P450 functions. Other regions show greater sequence divergence regions and are likely responsible for different substrate specificities between 2C members.
Several drugs for treating cardiovascular and psychiatric disorders are known substrates of cytochrome P450 2D6. (Dahl and Bertilsson, Pharmacogenetics 3, 61–70 (1993)), a situation that creates problems in prescribing such drugs. Although such drugs may be the most effective treatment for most of the population, physicians are reluctant to prescribe them due to the risk of adverse effects in poor metabolizers. Buchert et al., Pharmacogenetics 2, 2–11 (1992); Dahl et al., Pharmacogenetics 3, 61–70 (1993).
A complication in patient drug choice is that most drugs have not been characterized for their metabolism cytochromes P450. Without knowing which cytochrome(s) p450 is/are responsible for metabolizing an individual drug, an assessment cannot be made for the adequacy of a patient's P450 profile. For such drugs, there is a risk of adverse effects if the drugs are administered to deficient metabolizers.
The use of in vitro metabolism of therapeutic agents to address the potential in vivo induction, inhibition, drug-drug interaction and individual variability issues is known (for a recent review, see Rodrigues, 1994, Biochem. Pharmacol. 48: 2147–2156). Central to these studies is the unambiguous identification of specific drug-metabolizing enzyme(s), particularly human cytochrome P450 isoform(s) responsible for the metabolism of drugs. This objective can be achieved by using selective cytochrome P450 inhibitors, antibodies, recombinant cytochrome P450s and correlation analysis (Rodrigues, 1994, Biochem. Pharmacol. 48: 2147–2156).
Tripterygium Wilfordii Hook. F. (TW) is a native plant in China. Roots of plant Tripterygium Wilfordii Hook. F. contains bioactive components, primarily alkaloids, diterpenes and triterpenes. Historically, TW plant has been widely used in China to treat a variety of human diseases including autoimmune and/or inflammatory diseases for centuries. Studies have shown that diterpenes are major effective components in treating rheumatoid arthritis, chronic nephritis and some other diseases. However, there has been no study whatsoever on activities of each isolated diterpene compound nor any combinations thereof in human.
While the Tripterygium Wilfordii Hook. F. extract prepared according to the traditional method(s) has been used for treating autoimmune or inflammatory diseases for many years, each diterpene content in the preparations resulting from such method(s) varies from preparation to preparation and it has never been fully analyzed and quantified. Any attempt to quantify the major bioactive components has not been satisfactory so far due to the complexity of the extract composition and technical difficulties, where multiple compounds create great interference between the components among themselves. Hence, neither physicians nor patients have had informative knowledge about the amount of active components administered to the patients, although the medicine has been used for many years. As a result of such inconsistency in the drug dosages it is difficult for physicians to monitor the treatments following prognosis of the diseases. The lack of a well defined dosage regimens also prevents this herbal medicine, that has been proven highly effective in treating autoimmune and inflammatory diseases, from being further studied for the benefits of the public at large.
Despite that various TW extracts containing diterpenes have been reported to be effective for the treatment of autoimmune and/or inflammatory diseases, but such TW extracts may be highly toxic. There has been death report resulting from administration of certain TW extract. Ttriptolide (T10) has been reported as being carcinogenic or a major component causing significant side effects, while triptriolide (T11), tripdiolide (T8) and tripchlorolide (T4) are demonstrated to be the components having the most favorable therapeutic indexes, i.e. high efficacy and low toxicity in TW extract.
Studies on inhibition of cytochrome P450 enzyme activities is clearly of therapeutic importance. The co-administration of the TW extracts with another drug may increase or decrease the plasma level of the other drug, therefore, directly affect the efficacy of the other drug. In some instances, inhibition of the metabolism of other drugs by the TW extracts may result in or reduce the production of certain carcinogenic substances in the body. Accordingly, it is important for both drug development and clinical use to determine which cytochrome P450 enzymes are interact with the TW extracts, since cytochrome P450 enzymes are directly related to the metabolisms of many drugs.
Insofar as applicants know, there has been no study relating to drug interactions between any forms of the TW extracts and any other drugs. Without the knowledge of the profile of the TW extract drug interactions, it would be unlikely that the TW extracts will be of any significance in clinical or therapeutic uses.
The present invention provides a profile of the drug interactions of the TW extracts by investigating the effects of a particular form of the extracts, AHT-323A botanical extract, on a series of cytochrome P450 enzymes.